User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/03

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Summary

  • Preparing 4x Urea loading buffer
    • 4x Wilfred´s Urea loading dye (10 mL)
    • 4.8g Urea (4.8 g)
    • 1.5224g Thiourea
    • 3.2mL stacking gel buffer (0.625M) Tris-HCl pH 6.8
    • 0.8g SDS
    • 0.3722g EDTA
    • 0.025g Xylene Cyanol
    • 0.025g Bromophenol Blue
    • 4mL glycerol
    • Up to 10 mL with ddH20
  • Glycerol might not be necessary due to the fact that the high Urea concentration will provide enough weight to the sample, SDS might also not be necessary.
  • Glycerol was left out due to volume limitation
  • SDS Did not seem to solubilize before adding the color reagents, afterwards this could not be checked
    • Before adding H2O to solubilize SDS I wanted to add the color reagents to be sure not to add to much volume
  • Sample RIPA lysate S0 used before was gone, so now sample from 15March2010 is used

Materials & Methods

Method

Biorad Precision Plus Protein Dual Color marker
#2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SamplexPrecision plus markerhTERT RIPAhTERT PotterHEK293 RIPAHiMark markerhTERT RIPAhTERT PotterHEK293 RIPAPrecision plus markerhTERT RIPAhTERT PotterHEK293 RIPAHiMark markerx

Notes

  • Marker and Samples were too light so glycerol had to be added, due to time issues too much glycerol (~50%)!

Results

  • WHAT?

Discussion

  • Perhaps the amount of glycerol prevented the sample to condense to a nice band, maybe we can try with normal running buffer so that glycerol does not need to be added


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